Process for preparing antigens



Patented July--28, 1931 v j UNITED STATES PATENT OFFICE IAcoB- II. scmrnn, or wAsI INeTon, DISTRICT or COLUMBIA, AssIeNoR To ARTHUR m. HYDE, ,AS SECRETARY or AGRICULTURE or THE UNITED STATES or AMERICA f rnocnss non PREPARING ANTIGENS 1% Drawing.

Application med November 25, 1930. Serial nth-198,108.

(mam UNDER THE ACT or mm 3, I883, as summer unit so, 1928; 370 0.6. 757

This application is made under the -act of March 3, 1883, 215: amended by the act of April,

' 30, 1928, and the invention herein described may be manufactured and used by or for the '5 Government for governmental purposes without the payment to me of any royalty thereon.

Myinvention relates to the processof. pre-.

' paring antigens for use in the diagnosis of animal diseases and the products thereby produced. p Y

Up to the present timethere may be said to be two methods of testing fowls for pullorum diseases.

The first of these is known as the tube method, which is carried out by adding to suspensions of Salmonella pullorum different dilutions of the blood of chickens. In the case of a positive reaction the bacteria in the mixtures of antigen and serum clump and in time settle, leaving a clear fluid above. In the case of a negative reaction the tube remains uniformly clouded. This method requires the collection of considerable amounts of blood, theseparation of the serum from the blood, the subsequent accurate dilution of the serum and the incubation of the test tubes in laboratories. v

' To overcome certain objections to the tube method there has been devised a rapid 80 method. The antigen as generally employed I consists of a heavy suspension of Salmonella pullorum in salt solution. A few drops of this heavy suspension are placed-on a glass plate and a. drop or two of properly-diluted chicken serum or of blood are added directly to the bacterial suspension. In the case of a positive reaction if a bright light is placed beneath the glass plate on which the test is made it will be seen that the bacteria in the m well preserved'so' as to prevent contamination and so as to insure that the which are usedare killed.

In my work I have developed an anti on which consists of a heavy suspension of almonella pullorum in salt solution to which is added formaldehyde solution in sufficient amount to kill the bacteria. To this heavy suspension of Salmonellapullorum I have addedcrystal violet, an intensely staining dye, in an amount sufiicient to stain the suspended bacteria a distinct blue or purple. If new a rapid test he carried out as described under the rapid method above, it is found that pullorum bacteria in the case of a positive reaction the bacteria collect in the form of blue or purplish clumps which are distinct, especially if the test is carried out on a glass plate with a white background.

The antigen prepared as I have prescribed does not seem to interfere with the agglutination reaction in any way nor doesjit cause ag-' glutinationwhen mixed with the blood of normal fowls. In the latter case, instead of the stained bacteria being arranged in definite clumps with clear spaces between them there appears a uniform faintly bluish film. Tests have shown that the pullorum organisms employed in the stained antigen are killed and therefore there is no danger of the spread of disease-through the antigen. It has also been found that the dye employed aids in the prevention of contamination by extraneous organisms.

I have prepared a heavy suspension of ,Salmonella pullorum in 0.85 per cent salt solution killed by the addition of one per cent formalin and heavily stained by the addition of 0.03 per cent crystal violet.

The use of stained antigens as described above is new and useful in the diagnosis of animal diseases. Such stained antigens are superior'to the colorless products in the following respects:

(1) They yield reactions of greater visibility and therefore they may be used in less concentrated and less costly form.

(2) Because of the antiseptic action of the dye itself the stained antigens aremore readily and more perfectly preserved by the com- 1111051 antiseptics, suchas phenol or formaldee. (3) The [reactions may be obtained on glass plates and dried for permanent record i'fdesired. l

(4) Because the bacteria are colored in the stained antigen, no special illumination is re uired' to read the results of a test.

25) The stained antigen shows little or no deterioration on standing, nor is it necessary to keep it under refrigeration.

d Other antigens ma be similarly stained, for example, the antlgen, used in the rap d I agglutination test for abortion disease 1n 1s cattle. v I I By my description above I do not wish to restrict my-invention to the use of crystal violet and formaldehyde only since stains and preservatives other than crystal violet and formaldehyde may also be used and found suitable.

Having claim as my invention:

1. A diagnostic product comprising a heavy suspension of bacteria belonging to a disease producing class, anorganic dye, suchas is employed in staining bacteria, contrasting in color with the material to be tested, in

lorum disease in fowls comprising a heavy suspension of Salmonella pullorum in a saline solution, crystal violet in amount sufficient'to heavil stain the Salmonella pul- Iorum, said pro uct possessing the property when mixed with blood or serum of fowls infected with pullorum disease of rapidly agglutinating into deeply stained clumps of bacteria which stand out in sharp contrast to the red blood cells, especially when viewed against a white back ound. Y JACglS M. SCHAFFER.

fully-described my discovery I amount sufiicient to heavily stain the bacteria, 4

an organic preservative which does not interferewith'the diagnostic test, said I roduct possessing the propert when mix with blood or serum, of rapidly agglutinating in the case of positive reactlons into deeply stained clum of bacteria which stand out in sharp cotrast to the material to be tested, especial- 1y when viewed against awhite background.

2. A diagnostic heavy suspension of acteria belonging to a. disease producing class, an organic dye, such as is employed in staining bacteria, contrastin amount 'sufiicient to heavily stain the bacproduct comprising a .ing in color with the material to be tested,

teri a said roduct possem'ng the roperty when mix with blood or serum, or rapidly into deeply stained clumps of bacteria which stand out 1n sharp contrast to the'material to be tested, especially when viewed againsta white background.

v '3. A diagnostic product for detecti pullorum disease in owls comprising a eavy suspension of Salmonella pullorum in a saline solution, crystal violet inamount sufficlent to heavily stainthe Sahnonella pul- I lorunr, formaldehyde'in suflicient amount to kill the Salmonella pullorum, said product possessing the property when mixed with blood or-s'erumof fowls infected with pul- I agglutinating in the case of positive reactlons 1 l'orum disease of rapidly agglutinating into deeply stained clumps of bacteria which stand out in sharp contrast to the red. blood cells, especially when viewed against a white background. I Y I ,4. A diagnostic product for detecting pul- 

